Fig. 3. Loss of Ntn4 in PyMT mice accelerates tumor onset, progression, and metastasis.

(A) Immunohistochemical (IHC) staining images (left) and quantification (right) of NTN4 in the late-stage tumors of PyMT;Ntn4^wt and PyMT;Ntn4^−/− mice. n = 3 for both groups. Scale bars, 20 μm. (B) Palpable tumor onset in PyMT;Ntn4^wt (n = 16), PyMT;Ntn4^+/− (n = 14), and PyMT;Ntn4^−/− (n = 18) mice. (C) Tumor growth in PyMT;Ntn4^wt, PyMT;Ntn4^+/−, and PyMT;Ntn4^−/− mice. n = 6 per group. (D) Whole-mount–stained fourth mammary gland tumors images (left) and quantification of tumor area (right) in 13-week-old PyMT;Ntn4^wt (n = 8) and PyMT;Ntn4^−/− (n = 9) mice. Scale bars, 1 mm. LN, inguinal lymph node. (E) Tumor multiplicity in PyMT;Ntn4^wt (n = 16), PyMT;Ntn4^+/− (n = 14), and PyMT;Ntn4^−/− (n = 18) mice. (F) Hematoxylin and eosin (H&E) and IHC staining images comparing the normal mammary gland to mammary glands with various stages of PyMT tumors. Scale bars, 20 μm. (G) Tumor stages of fourth mammary glands from 13-week-old PyMT;Ntn4^wt (n = 6) and PyMT;Ntn4^−/− mice (n = 7). (H) IHC staining images for Ki-67 and PR in different stages of PyMT tumors from 13-week-old mice. Scale bars, 20 μm. (I) Representative images of lung surface metastatic nodules (top) and H&E staining of lung sections (bottom) from PyMT;Ntn4^wt, PyMT;Ntn4^+/−, and PyMT;Ntn4^−/− mice. Scale bars, 1 mm. (J and K) The number of metastatic nodules (J) in PyMT;Ntn4^wt (n = 16), PyMT;Ntn4^+/− (n = 14), and PyMT;Ntn4^−/− (n = 18) mice and metastatic foci (K) (n = 10) was quantified at the endpoint (20 to 25 weeks of age). Data are represented as means ± SEM. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.